Kinesin motor modulators derived from the marine sponge Adocia

ABSTRACT

This invention provides novel compounds derived from a marine sponge, Adocia sp., that specifically modulat kinesin activity by targeting the kinesin motor domain and mimicking the activity a microtubule. The compounds act as potent anti-mitogens are useful in a wide variety of in vitro and in vivo applications.

CROSS-REFERENCE TO RELATED INVENTIONS

This is a Divisional of application Ser. No. 09/226,772, filed Jan. 6,1999, now U.S. Pat. No. 6,207,403, which is a Continuation-in-Part ofU.S. patent application Ser. No. 60/070,772, filed on Jan. 8, 1998,which is herein incorprated by reference in its entirety for allpurpose.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. GM35252, awarded by the National Institutes of Health. The Government ofthe United States of America may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Plants and animals have yielded a number of chemical molecules havinguseful biological activity (e.g., anti-tumor activity). Particularlyrich sources of biologically active chemicals are marine organisms,which comprise over half a million species. Marine organisms have beenfound to produce a variety of metabolic often having unprecedentedchemical structures.

In recent years, an increasing number of natural products extracted frommarine organisms have been reported to exhibit a variety of biologicalactivities such as antimicrobial, antiviral, antifungal and anticanceractivities. These include peptides, polyethers, alkaloids, prostanoids,and the like. Such compounds have been obtained from sponges,octocorals, algae, tunicates, nuclibranches, bryozoans and marinebacteria.

In particular, a number of anti-tumor and anti-fungal compounds havebeen extracted from marine life. For example, U.S. Pat. No. 4,729,996discloses anti-tumor imidazole ring compounds isolated from the marinesponges Teichaxinella morchella and Ptioocaulis walpersi. U.S. Pat. No.4,808,590 discloses nitrogen containing cyclic compounds isolated havingantiviral, anti-tumor, and antifungal properties, isolated from themarine sponge Theoneloa sp. Similarly, U.S. Pat. No. 4,866,084 disclosesbisindole alkaloids extracted from the marine sponge Spongosoritesruetzleri useful in treating certain classes of tumors, while U.S. Pat.No. 4,970,226 discloses bis-indole imidazole alkaloids and derivativesisolated from the marine sponge Spongosorites sp. which exhibit usefulanti-tumor and antimicrobial properties.

Marine sponges, in particular, have proven to be a rich resource forbiologically active compounds (see, e.g., Scheuer, P. J. (ed.)(1978-1983) Marine Natural Products, Chemical and BiologicalPerspectives Vol. I-V, Academic Press, New York; Faulkner (1977)Tetrahedron, 33: 1421; Faulkner (1984) Nat, Prod. Rep. 1: 551; Faulkner(1986) Nat, Prod. Rep. 3: 1; Faulkner (1987) Nat, Prod. Rep. 4: 539;Faulkner (1988) Nat, Prod. Rep. 5: 613; Faulkner (1990) Nat, Prod. Rep.7: 269; Faulkner (1991) Nat, Prod. Rep. 7: 269; Faulkner (1992) Nat,Prod. Rep. 9: 323; Faulkner (1993) Nat, Prod. Rep. 10: 497; Faulkner(1994) Nat, Prod. Rep. 11: 355; Faulkner (1954) Nat, Prod. Rep. 12: 223;Faulkner (1996) Nat, Prod. Rep. 13: 75; Faulkner (1997) Nat, Prod. Rep.14: 256; and Faulkner (1985) J. Am. Chem. Soc. 107: 4796-4798). Howeverthere exist literally thousands of species of marine sponges and theseorganisms are only beginning to be explored.

SUMMARY OF THE INVENTION

This invention provides novel compounds derived from a marine sponge,Haliclona (aka Adocia) sp., that specifically modulate (e.g., inhibit)kinesin activity by targeting the kinesin motor domain and mimicking theactivity a microtubule. It is believed this mode of kinesin motormodulation is previously unknown. Thus, it was also a discovery of thisinvention that the kinesin-microtubule interaction site is a usefultarget for small molecule modulators of kinesin motor activity.

Because the compounds were initially derived from the marine spongeHaliclona (Adocia) sp. they are referred to herein as Adocia compoundsor Adocia-derived compounds. Particularly preferred Adocia-derivedcompounds are adociasulfates. Thus, in one embodiment, this inventionprovides for the Adocia-derived compounds having the formulas shownherein, more preferably for the adociasulfate compounds having theformulas shown herein.

The Adocia-derived compounds are potent kinesin motor modulators thatappear to block kinesin binding of microtubules. The compounds arepotent anti-mitotic agents that are highly effective in vitro and invivo. Thus, in another embodiment, this invention provides compositionfor the in vivo modulation (e.g., inhibition) of kinesin motor activity(e.g., in a cell). The compositions typically comprise any of theAdocia-derived kinesin motor inhibitors described herein in combinationwith a pharmacologically acceptable excipient.

In another embodiment, this invention provides methods of modulating(e.g., inhibiting) kinesin motor activity in a cell. The methods involvecontacting the cell with one or more of the Adocia-derived kinesinmodulators described herein. The cell, although preferably a mammaliancell, need not be so limited. Other suitable cells include, but are notlimited to, fungal cells and microbial cells. The cell can be in vitroor in vivo. Where the method is practiced in a therapeutic context(e.g., to ameliorate the effects of a pathological conditioncharacterized by hyperproliferation of one or more cells) theAdocia-derived kinesin modulators are preferably administered in atherapeutically effective dose.

In still another embodiment, this invention provides methods of assayinga test compound for kinesin modulatory activity. The methods involvecontacting a microtubule and a kinesin motor (e.g. a kinesin motorprotein) with one of the Adocia-derived kinesin modulators describedherein and detecting a change in kinesin motor activity resulting fromthe contacting. In a particularly preferred embodiment, the method ispracticed with one of the kinesin modulators of Formulas I, and III-VI,more preferably with one of the kinesin modulators of Formulas I or III.The change in motor activity is preferably detected through a motilityassay, a binding assay, an ADP release assay, or an assay foranti-mitotic activity. Typically the change in activity is evaluatedwith reference to a negative control (e.g., typically the same assay,but lacking a kinesin motor modulator) and/or with reference to apositive control (e.g., typically the same assay, with a differentkinesin motor inhibitor, preferably one whose activity has previouslybeen characterized).

This invention also provides Adocia-derived kinesin modulator kits. Thekits typically include a container containing one or more of theAdocia-derived kinesin modulator described herein. The kits canoptionally include a pharmacological excipient and/or a deliveryvehicle. When the excipient and/or delivery vehicle are provided theymay be provided combined with the kinesin motor inhibitor or in aseparate container for combination at the time of use. The kit can alsoinclude instructional materials describing the use of the compounds inany of the methods described herein.

In still another embodiment, this invention provides methods ofmodulating kinesin motor activity. The methods involve contacting thekinesin motor with a small organic molecule that competitively inhibitsthe kinesin motor at a microtubule binding site. in a particularlypreferred embodiment, the small organic molecule is an Adocia-derivedkinesin modulator as described herein or a a small organic molecule isidentified according to the methods described herein.

This invention also provides methods of identifying an agent thatmodulates the kinesin inhibitory activity of an Adocia kinesin inhibitor(e.g., on of the Adocia sulfates or Adocia derived kinesin inhibitorsdescribed herein). The methods involve contacting a microtubule and/or akinesin motor and/or an Adocia kinesin inhibitor with a candidate agent;and detecting a change in the kinesin inhibitory activity of the Adociakinesin inhibitor resulting from the contacting, wherein a changeindicates the identification of an agent that modulates the kinesininhibitory activity of the Adocia kinesin inhibitor.

Methods are also provided for identifying an agent that interferes withthe binding of an Adocia kinesin inhibitor with a kinesin. These methodsinvolve contacting a kinesin and an Adocia kinesin inhibitor (e.g. anadocia sulfate or an adocia derived kinesin modulator described herein)with a candidate agent; and detecting a decrease in the binding of theAdocia kinesin inhibitor with the kinesin resulting from saidcontacting, wherein a decrease indicates the identification of an agentthat interferes with the binding of the Adocia kinesin inhibitor and thekinesin.

Also provided herein is a complex comprising an Adocia kinesin inhibitorand a kinesin.

Methods are also provided for modulating cellular growth in an organism(e.g., an animal or a plant). The methods preferably involveadministering to the organism a composition comprising apharmaceutically acceptable carrier any one or more of the compoundsdescribed herein (e.g. adocia sulfates or adocia-derived kinesinmodulators) in a quantity sufficient to alter said cellular growth in anorganism.

Definitions

The term “molecular motor” refers to cytoskeletal molecule(s) thatutilize chemical energy to produce mechanical force, and drive themotile properties of the cytoskeleton.

The terms “kinesin” and “kinesin superfamily” as used herein refer to asuperfamily of eucaryotic motor proteins used to transport a largevariety of cargoes along microtubule “tracks”. Members of the kinesinsuperfamily are believed to be essential for mitotic and meiotic spindleorganization, chromosome segregation, organelle and vesicle transportand many other processes that require microtubule based transport. Thecommon feature of kinesins in the presence of a conserved ˜350 aminoacid motor domain which harbors the microtubule binding,ATP-hydrolyzing, and force transducing activities (see, e.g., Barton etal. (1996) Proc. Natl. Acad. Sci. USA, 93(5): 1735-1742, and Goldstein,(1993) Annu. Rev. Genet., 27: 319-351).

The term “kinesin motor” is used to refer to one or more proteinsinvolved in the transduction of chemical energy into mechanical energy.Kinesin is a force generating enzyme that hydrolyzes ATP to ADP andP_(i) and uses the derived chemical energy to induce plus end directedmovement along microtubules. This ubiquitous microtubule motor isthought to power anterograde organelle transport along microtubules. Theterm kinesin motor is intended to include kinesin related proteinsinhibition of which inhibits kinesin motor activity. Kinesin heavy andlight chains have been cloned and sequenced from a number of speciesincluding, but not limited to Drosophila (GenBank M24441), squid opticlobe (GenBank J05258), sea urchin and human (GenBank X65873), and rat(M75146, M75147, M75148), and the like (see, e.g., Yang et al. (1989)Cell 56: 879-889, Wright et al. (1991) J. Cell. Biol., 113: 817-833,Navone et al. (1992) J. Cell. Biol., 117: 1263-1275, and Cyr et al.(1991) Proc. Natl. Acad. Sci. USA, 88: 10114-10118). In addition, thescientific literature is replete with detailed descriptions of kinesins(kinesin motors) and kinesin related proteins (see, e.g., Kreis and Vale(1993) Guidebook to the Cytoskeletal and Motor Proteins, OxfordUniversity Press, Oxford, Vale (1990) Curr. Opin. Cell. Biol. 2: 15-22;Vale (1987) Ann. rev. Cell. Biol., 3: 347-378; and references therein).

The terms “kinesin motor inhibitor” or “inhibition of kinesin motoractivity” refers to the decrease or elimination of kinesin/microtubulemediated transduction of chemical energy (e.g. as stored in ATP) intomechanical energy (e.g., force generation or movement). Such a decreasecan be measured directly, e.g., as in a motility assay, or alternativelycan be ascertained by the use of surrogate markers such as a decrease inthe ATPase activity of the kinesin protein, and/or a decrease in theaffinity and/or specificity of kinesin motor protein-microtubule bindinginteractions, and/or in a decrease in mitotic activity of a cell orcells. Conversely, a “kinesin motor agonist” or “upregulator of kinesinmotor activity” refers to the increase of kinesin/microtubule mediatedtransduction of chemical energy (e.g. as stored in ATP) into mechanicalenergy (e.g., force generation or movement).

An “Adocia-derived compound” or “Adocia-derived kinesin modulator” asused herein refers to any of the kinesin modulators described herein(see, e.g. Formulas I, III, IV, V, and VI). It will be appreciated, thatwhile the Adocia-derived compounds include natural products derived fromsponges (or other marine organisms) the term also contemplates analoguesof such compounds as described herein. The Adocia-derived compounds thusneed not exist as natural products and may be chemically synthesized denovo.

The term “test compound” refers to a compound whose anti-kinesin motoractivity it is desired to determine. Such test compounds may includevirtually any molecule or mixture of molecules, alone or in a suitablecarrier.

The term “detecting the binding” means assessing the amount of a givensecond component that binds to a given first component in the presenceand absence of a test composition. This process generally involves theability to assess the amount of the second component associated with aknown fixed amount of the first component at selected intervals aftercontacting the first and second components. This may be accomplishede.g., by attaching to the second component a molecule or functionalgroup that can be visualized or measured (e.g., a fluorescent moiety, aradioactive atom, a biotin that can be detected using labeled avidin) orby using ligands that specifically bind to the second component. Thelevel of binding is preferably detected quantitatively. Binding, or achange in binding is indicated at the first detectable level. A changein binding, which can be an increase or a decrease, or presence versusabsence, is preferably a change of at least about 10%, more preferablyby at least about 20%, still more preferably by at least about 50%,still even more preferably by at least about 75%, even more preferablyby at least about 150% or 200% and most preferably is a change of atleast about 2 to about 10 fold (e.g., as compared to a control).

The phrase “detecting a change in the kinesin inhibitory activity of heAdocia kinesin inhibitor resulting from said contacting” refers todetermining the presence or absence or quantifying the alteration inkinesin inhibitory activity caused by a particular candidate agent,assays for such determinations are further described herein. A change inactivity, which can be in increase or a decrease, or presence versusabsence, is preferably a change of at least about 10%, more preferablyby at least about 20%, still more preferably by at least about 50%,still even more preferably by at least about 75%, even more preferablyby at least about 150% or 200% and most preferably is a change of atleast about 2 to about 10 fold (e.g., as compared to a control).

The phrase “detecting a change in kinesin motor activity resulting fromsaid contacting” refers to determining the presence, absence orquantifying the alteration in kinesin motor activity caused by aparticular composition (e.g., a test compound). The detecting caninvolve any one or more of a variety of assays for kinesin motoractivity as described herein. A change in activity, which can be anincrease or a decrease, or presence versus absence, is preferably achange of at least about 10%, more preferably by at least about 20%,still more preferably by at least about 50%, still even more preferablyby at least about 75%, even more preferably by at least about 150% or200% and most preferably is a change of at least about 2 to about 10fold (e.g., as compared to a control).

The term “compound” as used herein refers to organic or inorganicmolecules. The term includes, but is not limited to polypeptides,proteins, glycoproteins (e.g. antibodies), nucleic acids,oligonucleotides, and inorganic molecules.

The term “small organic molecule”, as used herein, refers to a compoundthat is an organic molecules of a size comparable to those organicmolecules generally used in pharmaceuticals. The term excludesbiological macromolecules (e.g., proteins, nucleic acids, etc.).Preferred small organic molecules range in size up to about 5000 Da,more preferably up to 2000 Da, and most preferably up to about 1000 Da.

A “bioagricultural compound” as used herein refers to a chemical or to abiological compound that has utility in agriculture or in environmentaland functions to foster food or fiber crop or crop protection or yieldimprovement. For example, one such compound may serve as a herbicide toselectively control weeds, as a fungicide to control the spreading ofplant diseases, as n insecticide to ward off and/or destroy insect,mite, and other arthropod pests. In addition, one such compound maydemonstrate utility in seed treatment to improve the growth environmentof a germinating seed, seedling, ro young plant as a plant regulator oractivator. Other compounds can serve in environmental management suchas, for example, forest management.

By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides, andpeptides. The protein may be made of naturally occurring amino acids andpeptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline, and norleucine are considered amino acids for the purposesof this invention. “Amino acid” also includes imino acid residues suchas proline and hydroxyproline. The side chains may be in either the (R)or the (S) configuration. In the preferred embodiment, the amino acidsare in the (S) or L-configuration. If non-naturally occurring sidechains are used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 141 9), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111 :2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

The term “competitive inhibition” is used to refer to competitiveinhibition in accord with the Michaelis-Menton model of enzyme kinetics.Competitive inhibition is recognized experimentally because the percentinhibition at a fixed inhibitor concentration is decreased by increasingthe substrate concentration. At sufficiently high substrateconcentration, V_(max) can essentially be restored even in the presenceof the inhibitor. Conversely, “non-competitive inhibition” refers toinhibition that is not reversed by increasing the substrateconcentration.

The term “cell” is used to refer to any cell including, but not limitedto mammalian, fungal, microbial and invertebrate cells. Preferred cellsinclude tumor cells including, but not limited to, carcinomas, includingbreast, ovary, prostate, skin, and colon; brain cancers, includingmemingioma, glioma, oligodendroglioma, embryonic cancers; sarcomass;leukemias, and lymphomas. Preferred cells also include neurons.Particularly preferred neurons are those related to neurodegenerativediseases including Alzheimer's Disease, Parkinson's Disease,Huntington's Disease, Frontotemporal Dementias, and Amyotrophic LateralSclerosis. Preferred cells further include cells derived from thegastrointestinal system including esophagus, stomach, intestine,pancreas, liver, lung, heart, and vascular system as sell as cells fromthe central and peripheral nervous system, kidney, bladder, muscularsystem and the bone system.

“In vivo” refers to in the living body of an organism.

“In vitro” refers to outside the living body, such as, an artificialenvironment, for example, a test tube or a cell or tissue culture.

The term “modulate” as used herein refers to increaing or decreasing anactivity of a molecule. Thus, for example, a kinesin motor modulatoracts to increase or decrease (inhibit) kinesin motor activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1 b, and 1 c show structures of preferred adociasulfates.

FIG. 2 shows adociasulfate-mediated inhibition of the microtubulestimulated ATPase of kinesin. The graph shows the dependence of theapparent k_(cat) value of microtubule-stimulated kinesin ATPase in thepresence of varying adociasulfate concentrations.

FIG. 3 shows that adociasulfate prevents binding of kinesin tomicrotubules. Monomeric kinesin protein K5-351 (3.5 μM), waspreincubated with microtubules (3.6 μM), MgAMP-PNP (2 mM),.with orwithout 35 μM adociasulfate (FIG. 1a). After 10 min the mixture wascentrifuged at 80,000×g for 30 min, and supernatants (S) and pellets (P)were analyzed by SDS-PAGE.

FIGS. 4a and 4 b show that adociasulfate inhibition is competitive withmicrotubule, but not with ATP. ATP-concentration dependence wasdetermined by a coupled enzymatic assay with pyruvate kinase and lactatedehydrogenase monitoring changes in absorbance at 340 nm (Huang et al.(1994) J. Biol., Chem., 269: 16508-16511). FIG. 4a shows that theapparent K_(m) for microtubules depends linearly on adociasulfateconcentration. The intercept with the x-axis gives a Ki value of 0.8 μM.b. Apparent Km for ATP does not depend on AS concentration, only V_(max)is affected,

FIGS. 5a and 5 b show that adociasulfates induce a burst of ADP release.To determine the percent of ADP released from kinesin (Hackney (1994) J.Biol. Chem., 269: 16508-16511) 80 μM kinesin was preincubated withα-³²P-ATP at room temperature for 15 minutes and then stored on ice. 1μM aliquots of that mixture were diluted into 100 μM of “chase mix”containing 0.5 mg/ml pyruvate kinase, 2 mM phosphoenolpyruvate andvarying concentrations of aldociasulfate (FIG. 1a). At different timepoints, 5 μM aliquots of the chase mix were quenched in 100 μM of 1 MHCl/1 mM ATP/1 mM ADP. The amount of ADP that became accessible topyruvate kinase and was converted to ATP was determined by thin layerchromatography (TLC) on PEI-cellulose followed by phosphoimager(Molecular Dynamics) quantitation. FIG. 4a shows an example of theexperimental trace. FIG. 4b shows that the magnitude of the burst of ADPrelease depends on adociasulfate concentration.

FIG. 6 illustrates synthetic schemes for the production ofAdocia-derived kinesin inhibitors of this invention.

DETAILED DESCRIPTION

This invention provides a previously unknown class of specific enzymemodulators that act to modulate binding of kinesin motor proteins tomicrotubules and thereby alter kinesin motor activity. It is believedthat prior to this invention, no small organic molecule modulators (thatare not nucleotides or nucleotide analogues) of kinesin motors wereknown or even suspected to exist.

The kinesin motor modulators of this invention were initially derivedfrom the sponge Adocia sp. and thus are referred to herein as Adociacompounds or Adocia-derived kinesin modulators (e.g. kinesininhibitors). Specific preferred Adocia compounds are sulfates andconsequently referred to herein as adociasulfates (AS).

I. Kinesin Motor Modulators

A) Uses of Kinesin Motor Modulators

The kinesin motor modulators of this invention are useful in a widevariety of contexts. In particular, preferred modulators of hisinvention act to inhibit activity of kinesin mediated transport. Thekinesins (members of the kinesin superfamily) are implicated inmicrotubule-mediated transport activities. As such they participate in awide variety of activities including, but not limited to mitotic andmeiotic spindle organization, chromosome segregation, organelle andvesicle transport and many others processes that require microtubulebased transport.

Modulation (e.g. inhibition) of kinesin motors therefor has profoundeffect on cellular function acting, for example, to inhibit meiosisand/or mitosis, and consequently inhibiting cellular growth and/orproliferation, e.g. in vitro or in humans and other non-human animals.As powerful anti-mitotics or anti-mejotics, the kinesin inhibitors ofthis invention have a wide variety of uses, particularly in thetreatment (e.g., amelioration) of, e.g. human and veterinary,pathological conditions characterized by abnormal cell proliferation.Such conditions include, but are not limited to: fungal infections,abnormal stimulation of endothelial cells (e.g., atherosclerosis), solidtumors and tumor metastasis, benign tumors, for example, hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,vascular malfunctions (e.g., arterio-venous malformations), abnormalwound healing, inflammatory and immune disorders, Bechet's disease, goutor gouty arthritis, abnormal angiogenesis accompanying: rheumatoidarthritis, psoriasis, diabetic retinopathy, and other ocular angiogenicdiseases such as retinopathy of prematurity (retrolental fibroplasic),macular degeneration, corneal overgrowth, corneal graft rejection,neuroscular glaucoma, Oster Webber syndrome, and the like. In addition,it is expected the kinesin motor inhibitors of this invention are usefulin the treatment/mitigation of a number of neurodegenerative disorders.

The kinesin motor modulators of this invention also find use in theprevention and treatment of plant diseases caused by, for example, fungi(e.g., Plasmodiophora brassicae (club foot of crucifers), Synchytriumendobioticum (potato black wart disease), Plasmopara viticola (downymildew of grape), Phytophthora infestans (late blight of pato andtomato), etc.), nematodes, insects, mites, or other arthropod pests, orparasitic seed plants (e.g., witchweed (Striga asiatica), dwarfmistletoe (Arceuthobium), etc.). Accordingly, the kinesin motormodulators find use in bioagricultural and environmental managementsettings as herbicides, fungicides, pesticides, or insecticides. In apreferred embodiment, the kinesin motor modulator is administered toplants with a bioagriculturally acceptable carrier or exipient.

The kinesin motor modulators of this invention also have a variety of invitro uses as well. For example, they can be used to freeze cells in aparticular stage of the cell cycle for a variety of purposes (e.g., inthe preparation of samples for of histological examination), in theisolation of nucleic acids from a particular stage of the cell cycle,and so forth.

The kinesin motor modulators of the invention also find use in thediagnosis of human and veterinary diseases, conditions, or pathologiesassociated with abnormal kinesin superfamily function, for example,disease states or conditions associated with hypersensitivity orresistance to kinesin motor modulators.

The kinesin motor modulators of this invention show unique specificityof kinesin motor/microtubule interactions. They therefor provide novellead compounds for the development of highly specific inhibitors orupregulators for kinesin families and subfamilies, thus allowing forprecise chemical intervention. In addition, the ability of the Adociacompounds of this invention to mimic microtubules in kinesin motorbinding allow the creation of artificial kinesin tracks for use invarious kinesin/microtubule assays (e.g., motility assays). Because oftheir ability to modulate the activity of kinesin motors (the conversionof chemical energy to mechanical activity) the kinesin motor modulatorsof this invention are useful for the production of nano-switches andother nano-devices (e.g., nanometer scale micro machines).

B) Preferred Kinesin Motor Modulators

In a preferred embodiment, this invention provides Adocia-derivedkinesin motor modulators characterized by a compound according toFormula (I)

in which R¹ and R² are either independent monovalent moietiesindependently selected from the group of H, hemiterpenes, terpenemonomers and terpene oligomers such that at least one of R¹ and R² isnot H, or R¹ and R² are combined to form a single divalent terpenemoiety selected from the group consisting of hemiterpenes, terpenemonomers and terpene oligomers; X¹ and X² are the same or different andare anionic derivatives of an organic group, an inorganic group or agroup which is a combination of organic and inorganic groups; Y^(+n) isan organic or inorganic cation; m is either 1 or 2; and n is either 1 or2.

Terpenes are characterized as being made up of units of isoprene in ahead-to-tail orientation. The terpenes are further classified by thenumber of isoprene units in their carbon skeletons, as shown in Table 1.

TABLE 1 Terpene classification Isoprene Units Carbon AtomsClassification 1 5 hemiterpene 2 10 monoterpene 3 15 sesquiterpene 4 20diterpene 5 25 sesterterpene 6 30 triterpene 8 40 tetraterpene >8 >40polyterpene

As used herein, the general term “terpenoid” encompasses hemiterpenes,terpene monomers, terpene oligomers and meroterpenes. “Terpene monomers”refers to derivatized or underivatized terpenes consisting of ten carbonatoms. “Terpene oligomers” refers to derivatized or underivatizedterpenes having more than ten carbons. “Meroterpenes” refers to terpenesthat are directly attached to any aromatic ring. Terpenoids which arenamed with a prefix (e.g., hemiterpenes, triterpenes, etc.) include bothderivatized and underivatized analogs of these terpenoids.

In a preferred embodiment, the terpendoids are oligomeric. In a furtherpreferred embodiment, the terpene oligomers are members selected fromthe group of diterpenes, sesquiterpenes, triterpenes, sesterterpenes andtriterpenes. In a still further preferred embodiment, the terpenoid is atriterpene.

Derivatized terpenes include, for example, terpene alcohols, aldehydes,ketones, ethers and esters. Each of these terms is used in its normalart-accepted manner. In a presently preferred embodiment, the inventionprovides compounds which are terpene alcohols. In another presentlypreferred embodiment, the invention provides compounds which are terpeneethers. In still a further preferred embodiment, the ethers are cyclicethers.

The compounds of the invention can be linear terpenes or terpenes whichinclude within their structural framework one or more rings. In apreferred embodiment, the terpenes are polycyclic, preferably havingmore than two rings and more preferably having more than four rings. Aring can be saturated, can contain unsaturation or can be aromatic. Aring can have from four to seven members and can consist of only carbonatoms or carbon atoms in conjunction with heteroatoms. Presentlypreferred heteroatoms include nitrogen, oxygen and sulfur. In apreferred embodiment, the rings are composed entirely of carbon atoms.In another preferred embodiment, a ring contains one or more oxygenheteroatoms. In a still further preferred embodiment, a ring contains asingle oxygen atom.

A compound of the invention can have either one or two terpenoidsattached to the benzene nucleus. When two terpenoids are present theycan be identical or different. Both terpenoids can contain one or morecyclic structures within their framework, both can be linear or one canbe linear and the other can contain one or more cyclic structures withinits framework. In this embodiment, a terpenoid can be attached to thebenzene nucleus as a monovalent moiety or it can be fused to the benzenenucleus as a divalent moiety. In another preferred embodiment, thecompounds consist of one terpenoid attached to the benzene nucleus. In afurther preferred embodiment, the terpenoid has at least one cyclicstructure within its framework. In a preferred embodiment, the terpenoidis fused to the benzene nucleus.

The benzene nucleus is functionalized with two anionic groups. Theanionic groups can be either the same or different and they are derivedfrom acidic organic groups, acidic inorganic groups or groups whichcontain an acidic inorganic group tethered to an organic group.

Acidic organic groups are primarily derived from carboxylic acids andthiocarboxylic acids. The carboxylic acids are attached to the benzenering directly or through a hydrocarbon chain of between one and fivecarbon atoms. In preferred embodiments, the organic acid is attacheddirectly to the benzene nucleus (e.g., phenylformic or phenylthioformicacid) or through a one carbon spacer (e.g., phenylacetic orphenylthioacetic acid).

When a hydrocarbon chain is present, this chain can be substituted withgroups which have the effect of modulating the acidity of the acid.Thus, electronegative groups (e.g., F, Cl, Br, NO₂, etc.) attached tothe hydrocarbon chain increase the acidity of the attached acidic group.In an opposite manner, electropositive groups (e.g., alkyl, alkenyl)decrease the acidity. The hydrocarbon chain can be attached to thebenzene nucleus through a carbon atom or can be attached via aheteroatom such as oxygen (e.g., glycolic acid) Inorganic acidic groupsare derived from inorganic acids including, but not limited to,phosphoric acid, phosphonic acid, phosphinic acid, boronic acid,sulfuric acid, sulfonic acid, arsonic acid and the like. The acid isbound to the benzene nucleus by either the central atom of the acid, orthrough an oxygen atom to form an “inorganic ester.” Examples of thesetwo modes of attachment include, for example, phenylphosphinic acid andphenyl phosphate, respectively. In a presently preferred embodiment, theacid is a sulfur containing acid. In an further preferred embodiment theacid is bound to the benzene nucleus via an oxygen atom. In yet anotherpreferred embodiment, the acid is derived from sulfuric acid.

Acids which are derived from species consisting of organic radicals andinorganic acids include, for example, alkyl sulfuric acids, alkylphosphoric acids, alkyl phosphinic acids and the like.

The cations which are associated with the anionic groups are eitherorganic or inorganic cations. The cations can have either a ⁺1 or ⁺2charge. When a cation with a ⁺1 charge used, two cations will beassociated with the molecule. When a ⁺2 cation is used, only one cationis necessary.

Inorganic cations include ions of Groups 1-12. Preferred inorganiccations include, but are not limited to, the cations of Li, Na, K, Cs,Mg, Ca, Mn, Fe, Co, Ni, Cu and Zn. Further preferred inorganic cationsare the cations of Li, Na and K.

Organic cations include, for example, tetraalkyl ammonium salts. Theammonium salts of the present invention are monovalent (e.g., R₄N⁺Y⁻) ordivalent as illustrated by Formula (II).

(R⁴)₃N—(CH₂)_(t)—R³—(CH₂)_(s)—N(R⁴)₃  (II)

in which R³ is a C₁ to C₁₀ aryl, substituted aryl, alkyl or substitutedalkyl group and R⁴ is lower alkyl or substituted lower alkyl. Theletters s and t represent integers from 1 to 5 and can be the same ordifferent. When the ammonium salt is monovalent, the alkyl group thecharacteristics of the nitrogen substituents will be generally the sameas those discussed in the context of R⁴.

A named R group will generally have the structure which is recognized inthe art as corresponding to R groups having that name. For the purposesof illustration, representative R groups as enumerated above are definedherein. These definitions are intended to supplement and illustrate, notpreclude, the definitions known to those of skill in the art.

The term “alkyl” is used herein to refer to a branched or unbranched,saturated or unsaturated, monovalent hydrocarbon radical having from1-10 carbons and preferably, from 1-6 carbons. When the alkyl group hasfrom 1-6 carbon atoms, it is referred to as a “lower alkyl.” Suitablealkyl radicals include, for example, methyl, ethyl, n-propyl, i-propyl,2-propenyl (or allyl), n-butyl, t-butyl, i-butyl (or 2-methylpropyl),etc.

The term “substituted alkyl” refers to alkyl as just described includingone or more functional groups such as lower alkyl, aryl, acyl, halogen(i.e., alkylhalos, e.g., CF₃), hydroxy, amino, alkoxy, alkylamino,acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, both saturated andunsaturated cyclic hydrocarbons, heterocycles and the like. These groupsmay be attached to any carbon of the alkyl moiety.

The term “aryl” is used herein to refer to an aromatic substituent whichmay be a single aromatic ring or multiple aromatic rings which are fusedtogether, linked covalently, or linked to a common group such as amethylene or ethylene moiety. The common linking group may also be acarbonyl as in benzophenone. The aromatic ring(s) may include phenyl,naphthyl, biphenyl, diphenylmethyl and benzophenone among others.

“Substituted aryl” refers to aryl as just described including one ormore functional groups such as lower alkyl, acyl, halogen, alkylhalos(e.g., CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,mercapto and both saturated and unsaturated cyclic hydrocarbons whichare fused to the aromatic ring(s), linked covalently or linked to acommon group such as a methylene or ethylene moiety. The linking groupmay also be a carbonyl such as in cyclohexyl phenyl ketone.

In a presently preferred embodiment, the organic cation is a compoundaccording to Formula (II). In a further preferred embodiment, R³ isphenyl and R⁴ is lower alkyl. In another preferred embodiment, both R³and R⁴ are lower alkyl. In these embodiments, s and t are preferablybetween 1 and 3, more preferably 1.

In a further embodiment, the invention provides for compounds having astructure according to Formula (III):

in which R⁵ and R⁶ are either independent monovalent moietiesindependently selected from the group consisting of H, hemiterpenes,terpene monomers and terpene oligomers such that at least on of R⁵ andR⁶ is not H, or R⁵ and R⁶ are combined to form a single divalent moietyselected from the group consisting of hemiterpenes, terpene monomers andterpene oligomers. In a presently preferred embodiment, R⁵ and R⁶ arecombined to form a seven-membered cyclic ether. In another preferredembodiment, R⁵ is a terpenoid and R⁶ is H.

In another embodiment, the present invention provides for adociasulfatecompounds having a structure according to Formula (IV).

In an additional embodiment, the invention provides an adociasulfatecompound having a structure according to Formula (V).

In a still further embodiment, the present invention provides anadociasulfate compound having a structure according to Formula (VI).

II. Isolation and/or Synthesis of Adocia-derived Kinesin MotorModulators

The Adocia-derived kinesin motor modulators of this invention can becreated de novo according to standard methods of chemical synthesis.Alternatively, where the modulators are natural products, they can beisolated from the organisms in which they are produced according tostandard methods.

A) Chemical Synthesis of Adocia Compounds

Using the structures provided herein, de novo synthesis of the compoundsof this invention can be achieved using standard methods well known tothose of ordinary skill in the art. In addition, the compounds of FIGS.1a, 1 b, and 1 c, which can be isolated from sponges, as describedbelow, provide convenient substrates which can be modified to producethe other compounds described herein. De novo synthesis of terpenoidsand/or modification of terpenoids is routine and well known to those ofskill in the art (see, e.g., U.S. Pat. No. 5,596,127 Process for thecontinuous preparation of terpene esters; U.S. Pat. No. 5,399,724Acyclic terpene compound; U.S. Pat. No. 5,202,460 Terpene derivatives,their preparation and their use; U.S. Pat. No. 5,073,659 Process for thepreparation of terpenes; U.S. Pat. No. 4,623,747 Terpene diesters andprocess for preparing the same; U.S. Pat. No. 4,137,257 Terpenehydroxysulfonic acids and corresponding hydroxysulfonate salts; U.S.Pat. No. 4,029,649 Terpene aryl esters, and Ho, (1988) Carbocycleconstruction in terpene synthesis,” New York; Kaufman et al. (1995) J.Med. Chem. 38: 1437-45; Gould, (1995) J. Cell. Biochem. Suppl.22:139-144; and Szirmai et al.(1995) Bioorg. Med. Chem. 3:899-906.Typical synthesis schemes are provide in FIG. 6.

B) Purification of Adocia Compounds

The compounds of the present invention can also be isolated from naturalsources. The fields of natural products isolation and terpene synthesisare well developed. Methods of purifying terpenoids of natural originand elucidating their structures are known to those of skill in the art.

In a preferred embodiment, the Adocia-derived kinesin motor modulatorsare purified according to the method described in Example 1.

III. Assay of Adocia-derived Kinesin Motor Modulators for Activity

It will be appreciated that the different Adocia compounds of thisinvention may exhibit different levels of kinesin motor modulatoryactivity. Consequently, it is desirable to identify those Adocia-derivedkinesin modulators of this invention that exhibit the highest level ofactivity and/or those that show various optimum levels of activity.Thus, in one embodiment, this invention provides methods of assaying(screening) the Adocia-derived kinesin motor modulators for activity.The screening may involve detection of presence or absence of kinesinmotor modulatory activity or quantification of such activity. In apreferred embodiment, such quantification is relative to a controllacking any modulator and/or to a reference kinesin modulator compound.The reference compound may be an modulator of this invention or adifferent modulator. Thus, for example, in a preferred embodiment, thescreened compound will be scored as a strong inhibitor if it hasinhibitory activity equal to or greater than the compounds of FormulasIV, V, or VI, more preferably the compound of formula IV, in a motility,binding, ATPase, or anti-mitotic assay (e.g. the assays described in theExamples herein). Particularly preferred screened compounds exceed theinhibitory activity of the compounds of Formulas IV, V, or VI, mostpreferably Formula IV by a factor of at least 2, more preferably by afactor of at least 5, and most preferably by a factor of at least 10.

While many assays for kinesin modulation are known to those of ordinaryskill in the art, particularly preferred assays include motility assays,binding assays, and assays for anti-mitotic activity.

A) Motility Assays

Because the microtubule/kinesin motor system transduces chemical energyinto force generation and molecular movement, motility assays provide aconvenient means for assaying for modulators of the kinesin motorproteins; stronger motor modulators producing a greater increase ordecrease in motility or having a particular effect at lowerconcentration. Generally motility assays involve immobilizing onecomponent of the system (e.g, the kinesin motor or the microtubule) andthen detecting movement, or inhibition thereof, of the other component.Thus, for example, in a preferred embodiment, the microtubule will beimmobilized (e.g., attached to a solid substrate) and the movement ofthe kinesin motor molecule(s) will be visually detected. Typically themolecule that is to be detected is labeled (e.g., with a fluorescentlabel) to facilitate detection.

Methods of performing motility assays are well known to those of skillin the art (see, e.g., Hall, et. al. (1996), Biophys. J., 71: 3467-3476,Turner et al., 1996, Anal. Biochem. 242 (1): 20-5; Gittes et al., 1996,Biophys. J. 70 (1): 418-29; Shirakawa et al., 1995, J. Exp. Biol. 198:1809-15; Winkelmann et al., 1995, Biophys. J. 68: 2444-53; Winkelmann etal., 1995, Biophys. J. 68: 72S, and the like). In addition, a suitablemotility assay is described in Example 2.

B) Binding Assays

In addition to, or in alternative to, motility assays, binding assayscan also be used to assay (detect and/or quantify) modulation of kinesinmotor proteins. In binding assays, the ability of the putative kinesinmotor modulator to inhibit or increase binding of the kinesin motorprotein(s) to microtubules are assayed.

There are a wide variety of formats for binding assays. In oneembodiment, the microtubule or the motor protein is attached to a solidsupport. The corresponding motor protein or microtubule is thencontacted to the support in the presence of the modulator to be screenedand the amount of bound motor protein or microtubule is then detectedand/or quantified (for suitable binding assay formats, see copendingapplication U.S. Ser. No. 60/057,895, filed on Sep. 4, 1997.

Solution phase binding assays are also known to those of skill in theart. For example, in one embodiment, the binding assay is akinesin-microtubule cosedimentation assay (Example 1). In this assay,(pelleting) assay, kinesin binds to microtubules in the presence ofAMP-PNP, a non-hydrolysable analogue of ATP, and sediments to the bottomof the tube after centrifugation to form a pellet. The nonhydrolysableATP analogue permits kinesin-microtubule binding, but not release.Meanwhile, unbound kinesin remains in the supernatant. The negativecontrol (lacking modulator) has saturating amounts of kinesin bound tothe microtubules. In the presence of kinesin motor inhibitors such asthe adociasulfates described herein, most of the motor remains in thesupernatant.

In one preferred embodiment, the assay, involves

1) Adding PEM80 and DMSO(control)/modulator to tube and mix thoroughly;

2) Adding other components: Kinesin, microtubules, and 2 mM MgAMP-PNP;

3) Centrifuging the tubes (e.g., in Beckman 42.2 rotor at 25,000 g, 30minutes, 20° C.);

4. Analyzing the supernatant and pellets on 10% SDS-PAGE.

Methods of performing kinesin motor-microtubule binding assays can befound in copending application U.S. Ser. No. 60/057895 filed on Sep. 4,1997. For a general description of different formats for protein bindingassays, including competitive binding assays and direct binding assays,see Stites and A. Terr (1991) Basic and Clinical Immunology, 7thEdition; Maggio (1980) Enzyme Immunoassay, CRC Press, Boca Raton, Fla.;and Tijssen (1985) Practice and Theory of Enzyme Immunoassays, inLaboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, B. V. Amsterdam.

C) ATPase Assay

Kinesin motors are effective ATPases hydrolyzing ATP to ADP to provideenergy for force generation. By examining ADP release from kinesin inthe presence of varying concentrations of kinesin motor modulator (e.g.,adociasulfate), the activity of the kinesin motor modulator can bequantified. One such ADP release assay is described in Example 1. In onepreferred embodiment, the ATPase activity assay utilizes 0.3 M PCA(perchloric acid) and malachite green reagent (8.27 mM sodium molybdateII, 0.33 mM malachite green oxalate, and 0.8 mM Triton X-100). Toperform the assay, 10 μM of reaction is quenched in 90 μl of cold 0.3 MPCA. Phosphate standards are used so data can be converted to mMinorganic phosphate released.

When all reactions and standards have been quenched in PCA, 100 μl ofmalachite green reagent is added to the to relevant wells in e.g., amicrotiter plate. The mixture is developed for 10-15 minutes and theplate is read at an absorbance of 650 nm. If phosphate standards wereused, absorbance readings can be converted to mM Pi and plotted overtime.

D) Mitotic Activity Assays

Assays for mitotic activity typically involve contacting a cell (invitro or in vivo) the test compound and assaying its effect on theability of the cell proliferate. Alternatively, a mass of cells can becontacted with the compound and the rate of growth of the mass can bemeasured.

1) Anti-mitotic Activity in situ

In one preferred embodiment, anti-mitotic activity of the kinesin motormodulator of this invention can be assayed in situ by testing theability o the modulator to alter the proliferation of new blood vesselcells (angiogenesis). Such proliferation assays are well known to thoseof skill in the art. One suitable assay is the chick embryochorioallantoic membrane (CAM) assay described by Crum et al. (1985)Science 230:1375. See also, U.S. Pat. No. 5,001,116, which describes theCAM assay.

Briefly, fertilized chick embryos are removed from their shell on day 3or 4, and a methylcellulose disc containing a particular amount (e.g.,100 mg) of the compound to be screened is implanted on thechorioallantoic membrane. The embryo are examined 48 hours later and, ifa clear a vascular zone appears around the methylcellulose disc, thediameter of that zone is measured. Using this assay, a disk of theAdocia derived compound of this invention is expected to alter cellmitosis and the growth of new blood vessels after 48 hours. Inhibitionof normal blood vessel growth indicates that the Adocia-derived kinesinmodulator is an inhibitor of cell mitosis and angiogenesis.

2) Anti-mitotic Activity in vitro

In vitro assays for anti-mitotic activity are also well know to those ofskill in the art. Typically such assays involve contacting a cell (e.g.a cell in culture) with the compound that is to be assayed anddetermining the effect on cellular proliferation. The cell can be onethat proliferates at a normal (e.g. endogenous) rate, or, alternatively,can be a cell in which hyperproliferation has been stimulated.Measurement of cellular proliferation can be direct (e.g., a cell count)or indirect, e.g., through a surrogate marker such as rate ofincorporation of a labeled amino acid. Such assays are standard and wellknown to those of skill in the art. Descriptions of assays foranti-mitotic activity are found for example, in U.S. Pat. Nos. 5,620,687and 5,443,962.

3) Anti-mitotic Activity in vivo

In vivo assays for anti-mitotic activity are also well known to those ofskill in the art. Typically these assays involve administering the testcompound to a subject organism and then evaluating the effect of thecompound on a target tissue or organ. Preferred organisms are those inwhich a hyperproliferative tissue or organ is manifest. Such organismsare well known to those of skill in the art and include, for example,standard tumor models (e.g., tumors introduced into nude mice) (see,e.g., Sharkey et al. (1990) Cancer Res. 50: 828s-834s). Organisms havingother natural or induced pathological conditions characterized byabnormal cell proliferation are also suitable.

E) Identifying Specific Modulators of Microtubule Binding

As explained above, it was a discovery of this invention that smallorganic molecules can specifically modulat kinesin activity by targetingthe kinesin motor domain and mimicking the activity a microtubule. Themolecules thus act as competitive inhibitors for microtubule binding.This is a previously unknown mechanism of kinesin (or other motor, e.g.,myosin or dynein) inhibition. Thus, in one embodiment, this inventionprovides methods of identifying kinesin inhibitors that specificallyblock the microtubule binding site. It is also expected that some smallorganic molecules will facilitate interactions at the microtubulebinding site and similar assays can be used to identify such enhancersof kinesin motor activity.

Such specific blockers are characterized by the fact that they can becompetitively inhibited by, or competitively inhibit, binders of themicrotubule binding site, but not binders at the ATPase site. In oneembodiment, this invention therefor provides methods of identifyingcompounds, especially small organic molecules, that change kinesin motoractivity by partially or completely blocking the microtubule bindingsite. The methods involve screening the “test” compound's ability tocompetitively inhibit binding of a moiety (e.g., ATP or an ATP analogue)at the ATPase site and screening the same compound's ability tocompetitively inhibit binding of a moiety (e.g., a microtubule) at themicrotubule binding at the microtubule binding site.

A kinesin modulator that shows competitive inhibition at the microtubulebinding site, but not at the ATPase site is identified as an inhibitorthat specifically binds to the microtubule site.

Methods of identifying competitive inhibition are well known to those ofskill in the art. Briefly, in the classical Michaelis-Menton model ofenzyme kinetics, competitive inhibition is easily recognizedexperimentally because the percent inhibition at a fixed inhibitorconcentration is decreased by increasing the substrate concentration.Thus, where the compound competitively inhibits binding at themicrotubule binding site, increasing the microtubule concentration at afixed concentration of test compound can restore the original(inhibitor-free) maximal rate of reaction (V_(max)). Conversely, wherecompetition is non-competitive increasing the substrate concentrationwill not restore the minimal rate of reaction (Vmax). Assays forspecific inhibition at the microtubule binding site are illustrated inexample 1. For a detailed discussion of analysis of reaction kinetics torecognize competitive, noncompetitive and uncompetitive inhibition, see,e.g., Lehninger (1975) Biochemistry Worth Pub., Inc. New York, N.Y.

Included herein are assays that identify agents which modulate theinteraction of Adocia kinesin modulators with kinesins. In the casewhere an agent is identified as altering (e.g., interfering with) thebinding of the inhibitor with the kinesin, that agent can be subjectedto further screening to determine its kinesin modulatory activity inaccordance with the assays provided herein. It is understood thatmicrotubules or their components are excluded as interfering agents.Similarly, identified modulators of the inhibitory activity of theAdocia kinesin inhibitors can be subjected to further screening. Inparticular, screens can be performed to determine whether the modulatorswork in conjunction or competitively with the Adocia kinesin inhibitors.Those modulators which function competitively are then further assayedindependently to determine their activity.

F) High Throughput Screening

Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. However, the current trend is to shorten the time scale forall aspects of drug discovery. Because of the ability to test largenumbers quickly and efficiently, high throughput screening (HTS) methodsare replacing conventional lead compound identification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of potential therapeuticcompounds (candidate compounds). Such “combinatorial chemical libraries”are then screened in one or more assays, as described herein, toidentify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics. In apreferred embodiment, combinatorial chemical libraries are providedcontaining the Adocia-derived compounds described herein.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationlike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. (Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,Hewlett-Packard, Palo Alto, Calif.) which mimic the manual syntheticoperations performed by a chemist. Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodification to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa. , Martek Biosciences, Columbia, Md., etc.).

Any of the assays for anti-kinesin motor activity described herein areamenable to high throughput screening. As described above, theadocia-derived compounds are preferably screened for anti-kinesin motoractivity in binding assays, motility assays, or assays for anti-mitoticactivity.

High throughput systems for such screening are well known to those ofskill in the art. Thus, for example, U.S. Pat. 5,559,410 discloses highthroughput screening methods for protein binding, while U.S. Pat. Nos.5,576,220 and 5,541,061 disclose high throughput methods of screeningfor ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configuarablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols the various high throughput. Thus,for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

IV. Modulation of Kinesin Motor Activity of Cells

In one embodiment, this invention provides for methods of modulatingkinesin motor activity of cells. It will be appreciated that were thecells are cells that hyperproliferate in vivo in any of the pathologicalconditions described above, the kinesin motor modulators of thisinvention can act as potent anti-mitotic therapeutic agents. However, itwill also be appreciated that therapeutic activity is not required forall uses of the compounds of this invention. The compounds can act assignificant lead compounds for the development of therapeutics.Alternatively, the compounds can be used to inhibit growth orproliferation of cells in vitro, to prevent contamination of biologicalsamples and the like with pathogenic organisms (e.g., fungi) or tofacilitate processing of the biological materials (e.g., in histologicalpreparations).

A) Indications

As indicated above, the kinesin motor modulators of this invention canbe used to mitigate a variety of pathological conditions (e.g., inhumans and animals) characterized by abnormal cell mitosis. Suchdiseases include, but are not limited to: abnormal stimulation ofendothelial cells (e.g., atherosclerosis), solid tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas, vascularmalfunctions, abnormal wound healing, inflammatory and immune disorders,Bechet's disease, gout or gouty arthritis, abnormal angiogenesisaccompanying: rheumatoid arthritis, psoriasis, diabetic retinopathy, andother ocular angiogenic diseases such as, macular degeneration, cornealgraft rejection, corneal overgrowth, neuroscular glacoma, Oster Webbersyndrome, and the like.

The kinesin motor modulators of this invention can also be used tomitigate variety plant diseases caused by abnormal cell division, orintracellular transport, or parasitic infections caused by, for example,microorganisms, nematodes, insects, and parasitic plants.

In addition, the kinesin motor modulators can be administered in vivofor non-therapeutic purposes (e.g., to rapidly kill and fix cells forhistological procedures), to elucidate the role of kinesin motors inearly development, and so forth. These applications are not intended tobe limiting, but rather indicative of the multiplicity of different usesfor the kinesin motor modulators of this invention. Other uses of thekinesin motors will be apparent to those of skill in the art.

B) Compositions for in vivo Administration

The kinesin motor modulators of this invention can be administeredorally, transdermally, by subcutaneous or other (e.g., intravenousinjection, intra-arterial injection, or direct injection into the targettissue) injection, intravenously, topically, parenterally,transdermally, or rectally. In addition, the kinesin motor modulatorsmay be incorporated into biodegradable polymers (or other reservoir)allowing for sustained release, the polymers being implanted in thevicinity of where delivery is desired, for example, at the site of atumor. The biodegradable polymers and their use are described in detailin Brem et al., (1991) J. Neurosurg. 74:441-446. The form in which thekinesin motor modulator will be administered (e.g., powder, tablet,capsule, solution, emulsion) will depend on the route by which it isadministered. The quantity of the drug to be administered will bedetermined on an individual basis, and will be based at least in part onconsideration of the individual's size, the severity of the symptoms tobe treated and the result sought as described above.

The kinesin motor modulator compounds are preferably ad ministered inthe form of an acid addition salt thereof, sequentially orsimultaneously with a pharmaceutically-acceptable carrier or diluent,especially and preferably in the form of a pharmaceutical compositionthereof, whether by the topical, oral, rectal, or parenteral (includingsubcutaneous) route, in an effective amount.

The compositions for administration will commonly comprise a solution ofthe kinesin motor modulator dissolved or suspended in a pharmaceuticallyacceptable carrier. A variety of carriers can be used, e.g., bufferedsaline containing suitable emulsifiers, and the like. Methods ofproducing liposomes and complexing or encapsulating compounds thereinare well known to those of skill in the art (see, e.g., Debs and Zhu(1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques6(7): 682-691; Rose U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309;and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414).

It is recognized, however, that the kinesin motor modulators of thisinvention are relatively charged molecules. Both charge and moleculesize tend to decrease cellular uptake and serum half-life. Consequently,in a preferred embodiment, it is desirable to package, complex, orotherwise combine the kinesin motor modulator with a delivery vehiclethat preferably increases cellular uptake and/or serum half-life.

A wide variety of suitable vehicles are well known to those of skill.Thus, for example, the kinesin motor modulator can be complexed with, orencapsulated within, a charged lipid to form a net neutral composition.This will reduce clearance by the reticuloendothelial system and enhancecellular uptake.

In another embodiment, the kinesin motor modulators can be encapsulatedwithin or complexed with microparticles which can be recognized andphagocytosed by a target cell thereby facilitating entry of the kinesinmotor modulator into the cell. Other methods of facilitating entryinclude the use of fusion proteins, protein complexes, and maskingcharged sulfate groups with reversible chemical modification orcounterions.

The size of particles and their mode of delivery determines theirbiological behavior. Strand et al. (1998) in Microspheres-BiomedicalApplications, A. Rembaum, ed., pp 193-227, CRC Press have described thefate of particles to be dependent on their size. Particles in the sizerange of a few nanometers (nm) to 100 nm enter the lymphatic capillariesfollowing interstitial injection, and phagocytosis may occur within thelymph nodes. After intravenous/intraarterial injection, particles lessthan about 2 microns will be rapidly cleared from the blood stream bythe reticuloendothelial system (RES), also known as the mononuclearphagocyte system (NPS). Particles larger than about 7 microns will,after intravenous injection, be trapped in the lung capillaries. Afterintraarterial injection, particles are trapped in the first capillarybed reached. Inhaled particles are trapped by the alveolar macrophages.It will also be appreciated that microparticles, an other deliveryvehicles can be targeted to specific cells and/or tissues (e.g., byconjugation with antibodies, or other cell or tissue specific ligands)or by the use of vehicles that have specific cell or tissue trophisms.

While the kinesin motor modulators of this invention are generally watersoluble, some species are moderately insoluble. Those compounds that arewater-insoluble or poorly water-are not well suited to conventionaladministration (e.g., by intravenous injection or oral administration).The parenteral administration of such pharmaceuticals can be achieved byemulsification of oil or lipid solubilized compound with an aqueousliquid (such as normal saline) in the presence of surfactants oremulsion stabilizers to produce stable microemulsions. These emulsionsmay be injected intravenously, provided the components of the emulsionare pharmacologically inert. For example, U.S. Pat. No. 4,073,943describes the administration of water-insoluble pharmacologically activeagents dissolved in oils and emulsified with water in the presence ofsurfactants such as egg phosphatides, pluronics (copolymers ofpolypropylene glycol and polyethylene glycol), polyglycerol oleate, etc.PCT International Publication No. WO85/00011 describes pharmaceuticalmicrodroplets of an anaesthetic coated with a phospholipid, such asdimyristoyl phosphatidylcholine, having suitable dimensions forintradermal or intravenous injection.

Additionally, protein microspheres have been utilized as carriers ofpharmacological or diagnostic agents. Microspheres of albumin have beenprepared by either heat denaturation or chemical crosslinking. Heatdenatured microspheres are produced from an emulsified mixture (e.g.,albumin, the agent to be incorporated, and a suitable oil) attemperatures between 100° C. and 150° C. The microspheres are thenwashed with a suitable solvent and stored. Leucuta et al. (1988)International Journal of Pharmaceutics 41: 213-217, describe the methodof preparation of heat denatured microspheres.

For certain of the therapeutic uses of the subject kinesin motormodulators, particularly cutaneous uses such as for the control ofkeratinocyte proliferation, direct (e.g., topical or injected)administration of the kinesin motor modulator will be appropriate.Accordingly, the subject kinesin motor modulator, alone or incombination with a delivery vehicle, may be conveniently formulated foradministration with a biologically acceptable medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Inpreferred embodiments, the kinesin motor modulator is dispersed in lipidformulations, such as miscelles, which closely resemble the lipidcomposition of natural cell membranes to which the kinesin motormodulator is to be delivered.

As indicated above, the kinesin motor modulators are preferably combinedwith a pharmaceutically acceptable carrier for in vivo administration.Pharmaceutically acceptable carriers (excipients) can contain aphysiologically acceptable compound that acts, for example, tosolubilize the composition, and/or to stabilize the composition, and/orto increase or decrease the absorption of the agent. Physiologicallyacceptable compounds can include, for example, carbohydrates, such asglucose, sucrose, or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low and/or high molecular weightproteins, compositions that reduce the clearance or hydrolysis of thekinesin motor modulator(s), or excipients or other stabilizers and/orbuffers. Other physiologically acceptable compounds include wettingagents, emulsifying agents, dispersing agents or preservatives which areparticularly useful for preventing the growth or action ofmicroorganism.

For those kinesin motor modulators that are lipid soluble the use ofsolubilizers and/or emulsifiers is often desired to produce aqueouskinesin motor modulator solutions or emulsions. Such solubilizers andemulsifiers are well known to those of skill in the art.

For example, lower alkyl alcohols having from 2 to 3 carbon atoms areuseful as diluents or solvents for kinesin motor modulators in thepreparation of stabilized kinesin motor modulator compositions of theinvention. Particularly useful alcohols are selected from the groupconsisting of ethyl alcohol, n-propyl alcohol and mixtures thereof.These alcohols are useful generally in the proportions by weight ofabout 1 to about 25 percent, preferably about 3 to about 15 percent,more preferably about 4 to about 10 percent, and most preferably about 4to about 6 percent by weight, all based upon the weight of the kinesinmotor modulator. These alcohols are miscible in both water and many oilsand can, therefore, be utilized as solvents for most of the forms of thefat-soluble kinesin motor modulators. These alcohols also serve tocontrol the viscosity of the kinesin motor modulator composition and actas secondary emulsifiers. Additionally, the alcohols can act as freezedepressants maintaining the fluidity of the kinesin motor modulatorcomposition at lower temperatures.

The emulsifier system optionally utilized in the kinesin motor modulatorcompositions of this invention can be selected from the various ionic ornonionic emulsifiers. The emulsifiers used must be acceptable asadditives for oral administration and/or for intravenous administrationand have no significant deleterious effect upon the kinesin motormodulator used therewith or upon the effectiveness of the lower alkylalcohol utilized as a solvent or diluent.

The kinesin motor modulator based pharmacological compositions arepreferably sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely divided active component. In, tablets, the activecomponent is mixed with the carrier having the necessary bindingcapacity in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain from five or ten to aboutseventy percent of the active compound. Suitable carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as carrier providing acapsule in which the active component, with or without carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid formssuitable for oral administration.

It is recognized that the kinesin motor modulators, when administeredorally, must be protected from digestion. This is typically accomplishedeither by complexing the kinesin motor modulator with a composition torender it resistant to acidic and enzymatic hydrolysis or by packagingthe kinesin motor modulator in an appropriately resistant carrier suchas a liposome. Means of protecting compounds from digestion are wellknown in the art (see, e.g., U.S. Pat. No. 5,391,377 describing lipidcompositions for oral delivery of therapeutic agents).

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogenous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water propylene glycol solutions. For example,parenteral injection liquid preparations can be formulated in solutionsin aqueous polyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizing and thickening agents, as desired.

Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The concentration of kinesin motor modulators or other activeingredients in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe patient's needs.

C) Dosages

Where the kinesin motor modulator is used in a therapeutic context(e.g., in the treatment of a condition characterized by cellularhyperproliferation), a therapeutically effective quantity ofadocia-derived kinesin modulator is employed in treatment. Atherapeutically effective quantity or dosage refers to a dosage adequateto ameliorate symptoms or signs of the disease or to provide effectiveprophylaxis without producing unacceptable toxicity to the patient. Ingeneral, an effective amount of the compound is that which provideseither subjective relief of symptoms or an objectively identifiableimprovement as noted by the clinician or other qualified observer.

The dosage of compounds used in accordance with this invention variesdepending on the compound and the condition being treated. The age,weight, and clinical condition of the recipient patient; and theexperience and judgment of the clinician, practitioner, or veterinarianadministering the therapy are among the factors affecting the selecteddosage. Other factors include the route of administration the patient,the patient's medical history, the severity of the disease process, andthe potency of the particular compound.

Broadly, a dosing schedule is from about 2 mg to about 2000 mg two orthree times a day. More typically, a dose is about 20 mg to about 400 mgof compound given three times a day.

A dosage range for topical treatment is about 0.1% to about 10%(weight/volume) in a physiologically acceptable eye drop applied one tofive or even ten times a day.

It will be appreciated that such dosages are typically advisorial innature and may be adjusted depending on the particular therapeuticcontext, patient tolerance, etc. Substantially higher dosages arepossible by any selected route, for example, topical administration.

Typically, the dosage is administered at least once a day until atherapeutic or prophylactic result is achieved. Preferably, the dosageis administered twice a day, but more or less frequent dosing can berecommended by the clinician. Once a therapeutic result is achieved, thedrug level can be modified for maintenance treatment. Under someconditions, the drug may be tapered or discontinued after the appearanceof a therapeutic result. Occasionally, side effects warrantdiscontinuation of therapy.

V. Adocia-derived Kinesin Motor Modulator Kits

In another embodiment, this invention provides kits for the practice ofthe methods of this invention. The kits preferably include one or morecontainers containing a adocia-derived kinesin motor modulator of thisinvention. The kit can optionally include a pharmaceutically acceptableexcipient and/or a delivery vehicle (e.g., a liposome). The kinesinmodulator may be provided suspended in the excipient and/or deliveryvehicle or may be provided as a separate component which can be latercombined with the excipient and/or delivery vehicle. The kit mayoptionally contain additional therapeutics to be co-administered withthe kinesin motor modulator.

The kits may also optionally include appropriate systems (e.g. opaquecontainers) or stabilizers (e.g. antioxidants) to prevent degradation ofthe kinesin motor modulators by light or other adverse conditions.

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of a Adocia-derivedkinesin motor modulator in the treatment of a disease in a mammalwherein the disease is characterized by cellular hyperproliferation. Inparticular the disease can include any one or more of the disordersdescribed herein.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1

Isolation of Adociasulfates

The sponge Adocia (Haliclona) sp. (Collection # 95-100) was collected inPalau, Western Caroline Islands, and was quickly frozen. The frozensponge (225 g) was diced and steeped in a mixture of dichloromethane(300 mL) and methanol (1 L) for 24 h. The solids were removed byfiltration and the solution was reduced in volume to 300 mL andextracted with dichloromethane (2×200 mL).

The aqueous phase was lyophilized to yield a pale yellow powder. Thepowder (1.0 g) was chromatographed twice on a reversed phase C18Sep-Pak, using a gradient of 30% MeOH in H₂O to 100% methanol (MeOH) aseluant, to obtain pure fractions containing adociasulfate-1 (FIG. 1a)and adociasulfate-2 (FIG. 1b) and a mixed fraction containingadociasulfates (FIGS. 1a, 1 b, and 1 c). The mixed fraction wasseparated by reversed phase HPLC using 1:1 MeOH-H₂O as eluant. Purefractions were combined to obtain adociasulfate-1 (13.5 mg),adociasulfate-2 (14.1 mg) and adociasulfate-3 (3.3 mg).

Example 2

Screening and Identification of Adocia-derived Kinesin Modulators

Motility Assay

TI-γ (a kinesin superfamily member from the fungus Thermomyceslanuginosus) was adsorbed to a glass coverslip and supplemented with amixture of microtubules, 2 mM Mg-ATP, and sponge extracts in DMSO (5%final concentration). Motility was scored visually on a Zeiss Axioplanmicroscope sat up for DIC and fitted with an Argus 10 video processor(Hamamatsu).

Proteins

All kinetic and binding measurements were performed on a bacteriallyexpressed Drosophila kinesin heavy chain fragment containing amino acids5-351 of the wild type protein and a hexahistidine tag at theC-terminus. Protein was purified from the soluble fraction of IPTGinduced bacterial cells by a single round of affinity chromatography onNi-NTA-agarose (Qiagen), concentrated by microfiltration, and frozen insmall aliquots in liquid nitrogen.

Steady State Kinetics

Initial rate measurements were done at room temperature using amalachite green assay (Geladopoulos et al.(1991) Anal. Biochem., 192:112-116) modified to work in 96 well microtiter plates and scored on aplate reader at 650 nm. ATP concentration dependence and basal ATPaserate were determined by a coupled enzymatic assay with pyruvate kinaseand lactate dehydrogenase monitoring changes in absorbance at 340 nm.Phosphate standards (650 μM-7 μM) were included with each reading.

ATPase Assay (ADP Release)

The percent of ADP released from the enzyme was determined by themethods of Hackney (see, e.g., Hackney (1994) J. Biol. Chem., 2690:16508-16511). Briefly, 80 μM kinesin was preincubated with α-³²PATP atroom temperature for 15 min and than stored on ice. 1 μl aliquots ofthat mixture were diluted into 100 μl of “chase mix” containing 0.5mg/ml pyruvate kinase, 2 mM phosphoenalpyruvate, and varyingconcentrations of adociasulfate. At different time points 5 μl aliquotsof the chase mix were quenched in 100 μl 1 M HCV/1 mM ATP/1 mM ADP. Theamount of ADP that became accessible to pyruvate kinase and wasconverted to ATP was determined by a thin layer chromatography onPEI-cellulose followed by phosphoimager quantitation.

In vivo Assays

The effects of adociasulfate (FIG. 1a) injection in the earlypre-cellular blastoderm embryo of Drosophila melanogaster wereevaluated. Embryos were collected every 20 minutes, dechorionated, andprepared for injection (Santarnaria (1986) paged 159-174 in Drosophila,A Practical Approach, D. B. Roberts, Eds., IRL Press, Oxford). Theembryos were desiccated for 7 minutes and pressure injected with eitherthe adociasulfate solution in injection buffer (5 mM KCl, 100 mM sodiumphosphate pH 7.5), or with buffer alone as control. The injected volumewas less than 10% of the total embryonic volume. Sets of 20 embryos wereinjected in each batch and at least three such batches were injected foreach different concentration of adociasulfate and control. The embryoswere then allowed to develop for 20-30 minutes at room temperatureinside a moist chamber and were subsequently fixed, devitelinized andimmunostained for tubulin, and counter stained with 0.01 mg/ml DAPI(Ashburner, Drosophila, a Laboratory Manual, Cold Spring HarborLaboratory Press, New York. (1989)).

Results

Extracts from 268 marine sponges were initially tested for their abilityto disrupt normal behavior of microtubules in a gliding motility assay.This screening method allowed immediate distinction between substancesthat affected microtubule movement and those that caused microtubuledepolymerization or breakage. Active extracts from the initial screeningwere then tested for inhibition of the microtubule-stimulated kinesinATPase.

The most promising candidates were extracts from the sponge Adocia sp.In the motility assay, these extracts disrupted microtubule attachmentto the kinesin-coated surface, and totally abolished movement. Themicrotubule stimulated ATPase of kinesin was also completely inhibited.

Three active compounds in the extract were identified and isolated (seeFIGS. 1a, 1 b, and 1 c). These specific compounds are referred to hereinas adociasulfates while the generic compounds are referred to as Adociacompounds or Adocia kinesin modulators (e.g., inhibitors). The structureof the Adocia compounds or adociasulfates does not resemble that ofnucleotide triphosphates. This indicates that the Adocia structures aredifferent from known kinesin modulators. In addition, it is believedthat the activity spectrum of the Adocia compounds is narrower than thatof nucleotide triphosphates or analogues thereof.

To further investigate specificity, the adociasulfate of FIG. 1a wastested on a variety of ATPases using the ATPase activity assay describedabove. Of those tested, the only enzymes substantially inhibited byadociasulfate are members of kinesin superfamily (Table 2).

TABLE 2 Concentrations of adociasulfate causing 50% inhibition ofenzymatic activity. enzyme C50 rabbit kidney ATPase >136 μM*Apyrase >136 μM* Myosin II (EDTA) 75 μM CENP-E 10 μM^(D) K5-351^(B)  2μM^(E) K411^(C)  2 μM^(E) TI-λ  2 μM^(E) ncd — myosin — pyruvate kinase— *enzyme was not inhibited by 50% at the highest used inhibitorconcentration of 136 μM.; ^(A)EDTA activated ATPase; ^(B)constructcontaining amino acids 5-351 of Drosophila kinesin; ^(C)constructcontaining first 411 amino acids of Drosophila kinesin; ^(D)at 6 μMtubulin; ^(E)at 2 μM tubulin

The behavior observed in the motility assay indicated thatadociasulfates interfere with microtubule binding to the motor. This wastested by performing a kinesin-microtubule co-sedimentation assay in thepresence of a nonhydrolysable ATP analog, AMP-PNP, with or withoutadociasulfate (FIG. 3). Addition of adociasulfate abolished binding ofkinesin to microtubules under these conditions.

Consideration of the kinesin mechanochemical cycle suggests that theeffect on microtubule binding could be induced either by looking thekinesin in a weakly-binding state resembling the kinesin-ADPintermediate by adociasulfate binding in the nucleotide pocket, or bydirect interference with the microtubule-binding site. Steady statekinetic measurements demonstrated that the adociasulfate-inducedinhibition is competitive with microtubules, and could be totallyreversed by high microtubule concentrations (FIG. 4a).

In contrast, varying the ATP concentration had no effect on the overallshape of the kinetic curves. V_(max) was progressively lower at higheradociasulfate concentrations (FIG. 4b). An additional argument againstadociasulfate binding at the nucleotide pocket comes from the lack of aninhibitory effect on the basal, non microtubule-stimulated rate of thekinesin ATPases. If adociasulfate interfered with nucleotide binding, orlocked the enzyme in a particular nucleotide-bound state, ATP turnoverin the absence of microtubule should be decreased. However,concentrations of up to 136 μM adociasulfate (the highest tested) didnot inhibit the basal ATPase rate.

Microtubule binding to kinesin induced 1,000-fold stimulation of thebasal ATPase rate, owing primarily to accelerated ADP release. Is wastested whether adociasulfate binding to kinesin could mimic the effectof the microtubule by examining ADP release from kinesin in the presenceof varying concentrations of adociasulfate. Indeed, bursts of ADPrelease were observed and their magnitude correlated positively with theconcentration of adociasulfate (FIGS. 5a and 5 b). The adociasulfateconcentration at 50% of maximum burst is much higher than the K_(i)determined in steady state microtubule competition assays. Thisdiscrepancy may reflect different affinities for adociasulfate indifferent nucleotide states of kinesin. Steady state kineticmeasurements of K_(i) reflect the affinity of the most tightly boundstate of the entire cycle, which includes several kinesin-nucleotideintermediates (K-ATP, K-ADP-Pi, K-ADP etc.).

In contrast, the ADP release experiment with adociasulfate involved onlyone state, K-ADP. It is intriguing that this state also has the lowestaffinity for the microtubule. It was initially surprising thatadociasulfate did not stimulate the kinesin basal ATPase even though itinduced ADP release. However, during steady state kinetic measurements,each single headed kinesin molecule must undergo several cycles ofattachment-detachment to microtubule subunits. In contrast, ASpresumably remains bound through multiple enzymatic turnovers. Thephysiological equivalent of such a state would be a kinesin moleculepermanently attached to a single tubulin dimer, a state for which nokinetic data exist. However, if the adociasulfate binding to kinesinresembles microtubule binding, the initial association event shouldresult in a burst of ADP release as observed.

Example 3 In vivo Effects of Adociasulfates

The in vivo effects of adociasulfate were also investigated. Preliminaryexperiments demonstrated that AS had no effect on HeLa cellsproliferation. However, because it is a fairly large molecule (MW 738)with two charged sulfate moieties, adociasulfate may have problemscrossing the cell membrane. This difficulty can be alleviated by directinjection of adociasulfate into a cell.

Drosophila embryos, a well characterized and sturdy system, were thusused for direct injection studies. In Drosophila embryos the first 13rounds of cell division take place in a syncitium during the first twohours after fertilization. The first seven of these divisions aresynchronized and occur at the center of the egg yolk. During theremaining divisions, most nucleic move to the surface of the egg andcontinue to divide for the next three times in partially open cell buds(see, Campos-Ortega et al. (1985) The embryonic development ofDrosophila melanogaster, Springer Verlag). Thus, any drug injected in tothe Drosophila will have access to a large number of mitotic nuclei atthe surface of the egg during the last three divisions.

Three different concentrations of adociasulfate were injected (1 mM, 0.1mM, and 0.05 mM) in combination with 1 mg/ml tetramethylrhodamine(TMR-tubulin. Injection of up to 0.1 mM adociasulfate arrested allnuclear divisions immediately at the point of injection. Injection of0.05 mM adociasulfate caused less severe phenotypes that allowedobservation of more distinct abnormalities. Spindles and microtubuleasters without chromosomes were found at the site of injection. Mats ofunattached microtubules and chromosomes apparently detached from thespindle were also observed. These effects could be accounted for by lossof function of various members of the kinesin superfamily.

These results demonstrate that adociasulfate specifically modulatskinesin activity by interfering with microtubule binding. This mechanismis unlike that of any know kinesin (or other motor) inhibitor. Withoutbeing bound to a theory it is believed that adociasulfate modulatsbinding by emulating tubulin binding to a portion of the microtubulebinding site of kinesin. In addition, adociasulfate is a potent toxin,which, when delivered intracellularly, may ablate several, if not all,aspects of kinesin-superfamily mediated transport.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference.

What is claimed is:
 1. A method of modulating kinesin motor activity,said method comprising contacting said kinesin motor with a moleculethat competitively inhibits said kinesin motor at a microtubule bindingsite, wherein said molecule is identified according to the followingsteps: (i) assaying for competitive inhibition of said kinesin motoractivity by said molecule at a kinesin ATPase site; (ii) assaying forcompetitive inhibition of said kinesin motor activity by said moleculeat a microtubule binding site; and (iii) identifying said molecule as akinesin modulator specific to a microtubule binding site when saidmolecule is a competitive modulator at said microtubule binding site,but not at said ATPase site.
 2. The method of claim 1, wherein saidassaying comprises detecting ATPase activity of said kinesin motor. 3.The method of claim 1, wherein said molecule is a polypeptide.
 4. Themethod of claim 1, wherein said molecule is a nucleic acid.
 5. Themethod of claim 1, wherein said molecule is a small organic molecule. 6.The method of claim 1, further comprising the step of administering toan organism a composition comprising a pharmaceutically acceptablecarrier and said molecule in a quantity sufficient to alter saidcellular growth of said organism.
 7. The method of claim 6, wherein saidorganism is an animal.
 8. The method of claim 6, wherein said organismis a plant.
 9. The method of claim 1, wherein said molecule is a part ofa combinatorial chemical library.
 10. The method of claim 1, whereinsaid molecule has a terpene substituent.
 11. The method of claim 1,wherein said kinesin motor is at least one kinesin heavy chain.
 12. Themethod of claim 11, wherein said kinesin motor is a protein comprising asequence of amino acids 5 to 351 of wild-type Drosophila kinesin heavychain.
 13. The method of claim 11, wherein said kinesin motor is aprotein comprising a sequence of amino acids 1 to 411 of wild-typeDrosophila kinesin heavy chain.
 14. The method of claim 1, wherein saidkinesin motor is Thermomyces lanuginosus gamma kinesin-like protein. 15.The method of claim 1, wherein said kinesin motor is centromere bindingprotein E.
 16. The method of claim 1, wherein said kinesin motor is akinesin-related protein.
 17. The method of claim 1, wherein saidassaying comprises testing molecules in a gliding motility assay.